| ObjectiveIrritable Bowel Syndrome (IBS) is a clinical commonly encountered disease of digestive tract with high incidence and influences the life and work of patients. Part of patients got the symptoms of IBS after recovering from acute inflammation of gastrointestinal and it is called post inflammation irritable bowel syndrome (PI-IBS). The pathogenesis of PI-IBS is very complicated and still unclear, but the the regulation effects of brain-gut axis have a very important function im its mechanism.5-hydroxytryptamine (5-HT) is a kind of brain-gut petide which wildly distributed in central nerve system and gastrointestinal tract, participates in regulation of brain-gut axis and has fatal effects in the pathogenesis of PI-IBS. Wuji pill, which was constituted of huanglian, wuzhuyu and baishao, with the effects of Shuganlipi and Qingrehewei, was commonly used in treatment of IBS. In this study, Wuji pill with two different compatibilities were used as therapeutic drugs to deal with the rats of PI-IBS which was established by intracolonic instillation of acetic acid with restraint stress. The colon motility and the function of5-HT signal system in central fringe system and colon were observed to study the in vivo pharmacodynamics actin and mechanism of Wuji pill with two different compatibilities, then apply the ex vivo experiments with rat colon smooth muscle cells to explore the mechanism of Wuji pill with two different compatibilities in improving the colonic motility of PI-IBS, furthermore compared the similarities and differences of Wuji pill with two different compatibilities and provided foundations for compatibility theory of traditional chinese medicine and its clinical application.Methods and Results1Induction of PI-IBS rat modelMethod:The PI-IBS rat models were established by by intracolonic instillation of1ml4%acetic acid to induce acute inflammation of colon, after7days post-enema, the rats were wrap-restraint or immobilized with cold. Compared the colonic motility, contents of5-HT (in serum, colon, spinal cord and brain), quantity of mast cells and its degranulation rate in colon, in order to choose a suitable model for further study.Results:The colonic motility of the two models were significantly accelerated, but no differences between each other; the content of5-HT were remarkably increased in serum, colon and spinal cord but with a downtrend in brain, moreover the wrap-restraint group had notable difference with normal group; the quantity of mast cells was increased but had no differences and the degranulation rate in two model groups were much more higher than normal group, especially in the immobilized with cold group.Conclusion:The PI-IBS rat model established by intracolonic instillation acetic acid with wrap-restraint has advantages in decreasing the content of5-HT in brain, so it was chose to further study.2Study of pharmacodynamics of Wuji pill on the colonic motility of PI-IBSMotheds:PI-IBS rat model was established by intracolonic instillation of acetic acid with wrap-restraint stress, rats in each group were given Dicetel and different dose of Wuji pill of two compatibilities respectively after stress, and the rats in model and normal control group were given saline of same volume for7days. The colonic motility index, the number of feces defecated in2hours and the discharge time of glass beads were measured before enema, after stress and after treated for7days to evaluate the function of colon movement.Results:There were no differences in motility index (MI) between groups before enema. After stress, the MI and the change rate of MI in model groups were significantly increased(P<0.01). The middle-dose and high-dose of two compatibilities can remarkably decrease the MI and the change rate of MI(P<0.01). The effects of high-dose Wuji pill on motility index and high-dose NO.2Wuji pill were similar with Dicetel. There were notable differences between different dose and no differences between the same doses of two compatibilities.There were no differences in the discharge time of glass beads between groups before enema. After stress, the discharge time in model groups were obviously shorter than normal control group(P<0.01). The middle-dose and high-dose of two compatibilities can significantly prolong the discharge time(P<0.05, P<0.01). There were no differences between positive control group and high-dose Wuji pill groups. There were notable differences between different dose(P<0.01) and no differences between the same dose of two compatibilities.There were no statistical differences in the number of feces defecated in2hours between groups before enema. After stress, the number of feces defecated was notably increased compared with normal control group(P<0.01). The high-dose of two compatibilities can remarkably decreased the feces defecated number(P<0.05, P<0.01). The effects of middle-dose and high-dose Wuji pill were similar with Dicetel in reducing the feces number. There was obviously difference between low-dose and high-dose group of NO.2Wuji pill(P<0.01).3The mechanism study of Wuji pill on colonic motility of PI-IBSMethods:After treated for7days, all the rats were anaesthetized and then collected the blood, colon, hippocampus, hypothalamus and frontal lobe. Apply high performance liquid chromatography with electron capture detector to test the content of5-HT and immunohistochemistry stain to measure the5-HT positive expression, the result were investigated by qualitatively and quantitatively by means of image pro plus software through analyzing the opacity density(OD). Immunofluorescence stain was used to evaluate the5-HT receptor3and4in colon and hippocampus and the results were analyzed by laser scanning confocal microscope. The adenyl cyclase, phosphodiesterase4and calcium ion concentration in colon and hippocampus were tested by ELISA and the phosphorylation level of c AMP-response element binding protein was investigated by western blotting. All the data was expressed as mean±standard deviation.Results:The content of5-HT in serum of model group was increased but no difference with normal control group, the content of5-HIAA and the percent conversion of5-HT were significantly decreased(P<0.05, P<0.01). High-dose of NO.1Wuji pill can remarkably decrease the content of5-HT, middle-dose and high-dose of NO.1Wuji pill can notably increase the content of5-HIAA, the two compatibilities all can obviously improve the percent conversion of5-HT except low-dose of NO.2Wuji pill (P<0.05, P<0.01). The effects of high-dose of NO.2Wuji pill on increasing5-HIAA, middle-dose and high-dose of NO.2Wuji pill on improving the percent conversion of5-HT were better than the same dose of NO.1Wuji pill.The content of5-HT and5-HIAA in colon of model group were notably increased (P<0.01) and the percent conversion of5-HT was decreasing, but no difference was observed. Two compatibilities can remarkably decrease the content of5-HT, Middle-dose and High-dose can obviously decrease the5-HIAA(P<0.05, P<0.01) and no differences in percent conversion of5-HT. The two compatibilities had preponderance in High-dose compared with Dicetel, but no differences between the same doses of the two compatibilities.The5-HT positive expression in colon submucous nerve plexus and syenteric nerve plexus of model group was notably increased (P<0.01), two compatibilities can decrease it remarkably(P<0.01) and the effect was directly related to dose, but no statistical differences were observed between the same dose of two compatibilities.The fluorescence intensity of5-HT receptor3and4were remarkably increased in colon muscularis of model group (P<0.01), two compatibilities can decrease it notably(P<0.01) and the effect was directly related to dose. The effects of Middle-dose and High-dose of NO.1Wuji pill on reducing5-HT receptor3and Low-dose on reducing5-HT receptor4were better than NO.2Wuji pill (P<0.05, P <0.01).The calcium ion and the activity of adenyl cyclase were increased significantly and the phosphodiesterase4was obviously decreased (P<0.01) in colon of model group, Wuji pill had predominant effects on down regulating Ca2+, AC and up regulating PDE4(P<0.05, P<0.01). NO.2Wuji pill of Middle-dose and High-dose had the similar effects with Dicetel on down regulating Ca2+and AC, better effect on up regulating PDE4than Dicetel (P<0.01). Compared with the same dose of NO.1Wuji pill, NO.2had better effects on down regulating Ca2+, AC and Low-dose of NO.2can up regulating PDE4remarkably. There were no differences in expression of CREB in each group, but the phosphorylation level of CREB in model group were increased significantly (P<0.01). Wuji pill can obviously down regulate it (P<0.05,P<0.01) except Low-dose of NO.1Wuji pill, and there was no difference in the same dose of two compatibilities.The quantity of mast cells and enterochromaffin cells, the degranulation rate of mast cell were increased obviously (P<0.01) in colon of model group. The down regulating effects of Wuji pill were remarkable (P<0.01) and directly related to dose. High-dose of Wuji pill had the similar effect on decreasing the quantity of mast cell and enterochromaffin cells while the high-dose of NO.1Wuji pill also had no difference with Dicetel on down regulating the degranulation rate of mast cell and there was no difference in the same dose of two compatibilities.The content of5-HT was obviously decreased while the content of5-HIAA and the percent conversion of5-HT were increased significantly in hippocampus of model group (P<0.05, P<0.01). Each dose of Wuji pill can up regulate5-HT and the High-dose can down regulate5-HIAA notably while two compatibilities can decrease the percent conversion of5-HT significantly (P<0.05, P<0.01) except the Low-dose of NO.2Wuji pill, and the effects were directly related to dose. There was no difference in the same dose of two compatibilities.The content of5-HT and5-HIAA was obviously decreased(P<0.05, P<0.01) while the percent conversion of5-HT were increased significantly in hypothalamus of model group (P<0.01). Each dose of Wuji pill can up regulate5-HT and decrease the percent conversion of5-HT significantly except the Low-dose of NO.1Wuji pill while the Middle-dose of two compatibilities can up regulate5-HIAA notably (P<0.05, P<0.01). The effect of High-dose of NO.1Wuji pill on increasing the content of5-HT was better than Dicetel, but there was no difference in the same dose of two compatibilities.The content of5-HT was obviously decreased while the content of5-HIAA and the percent conversion of5-HT were increased significantly in frontal lobe of model group (P<0.01). Each dose of Wuji pill can down regulate5-HIAA while the Middle-dose and High-dose can up regulate5-HT notably and decrease the percent conversion of5-HT significantly (P<0.05, P<0.01). There was no difference in the same dose of two compatibilities.The positive expression of5-HT were decreased in hippocampus and hypothalamus while it was increased obviously in frontal lobe (P<0.01)in model group. Wuji pill can significantly increase it in hippocampus and hypothalamus, and also can decrease it in frontal lobe (P<0.05, P<0.01) except Low-dose of NO.1Wuji pill, and the effects were directly related to dose. The up regulation effects of Middle&High-dose and the down regulation effects of High-dose of NO.1Wuji pill and Middle&High-dose of NO.2Wuji pill were better than Dicetel (P<0.05, P<0.01). The up regulation effects of High-dose of NO.2Wuji pill in hypothalamus and the down regulation effects of Middle&High-dose of NO.2Wuji pill were better than the same dose of NO.1Wuji pill (P<0.05, P<0.01).The fluorescence intensity of5-HT receptor3and4were remarkably decreased in hippocampus of model group (P<0.01), two compatibilities can increase it notably(P<0.01) and the effect was directly related to dose. The effects of Middle&High-dose of NO.2and High-dose of NO.1Wuji pill on increasing5-HT receptor3&4were better than Dicetel (P<0.05, P<0.01). The effects of Low&Middle-dose of NO.2on increasing5-HT receptor3and Middle&High-dose of NO.2Wuji pill on increasing5-HT receptor4were better than the same dose of NO.1(P<0.05, P<0.01)The calcium ion and the phosphodiesterase4were increased significantly and the activity of adenyl cyclase was obviously decreased (P<0.01) in hippocampus of model group. Wuji pill had predominant effects on down regulating Ca2+PDE4and up regulating AC (P<0.05, P<0.01), and the effects were directly related to dose. Low&High-dose of NO.1Wuji pill had better effects on reducing Ca2+and High-dose of NO.1Wuji pill had better regulations on AC and PDE4than the same dose of NO.2Wuji pill (P<0.05).4Influence of Wuji pill on5-HT receptor3&4and downstream signaling pathway of rat colon smooth muscle cellsMethods:According to Bitar’s methods, the rat colon smooth muscle cells (SMC) were isolated and cultured, and then identified by a-actin immunohistochemistry stain. Furthermore, tested the5-HT receptor3&4of the SMC membrane and Ca2+by immunofluorescence stain and calcium ion probe to evaluate the influence of Wuji pill on the change of5-HT receptor3&4caused by agonist and antagonist of5-HT receptor3&4. The cAMP and PKA level were measured by ELISA to estimate the effect of Wuji pill on the change of cAMP and PKA caused by agonist and antagonist of5-HT receptor4and AC.Results:Two compatibilities can decrease the expression of5-HT receptor3&4and the concentration of Ca2+of the SMC significantly, furthermore inhibit the elevation of5-HT receptor3&4and Ca2+caused by phenylbiguanide and Tegaserod dramatically, also obviously promote the reduction of5-HT receptor3&4and Ca2+caused by Alosetron and GR113808. The effect of NO.2Wuji pill was better than NO.1.Two compatibilities can down regulate the level of cAMP and PKA of the SMC significantly, furthermore obviously inhibit the elevation of cAMP and PKA caused by Tegaserod and Forskolin, and also dramatically promote the reduction of cAMP and PKA caused by GRl13808and SQ22536. The effect of NO.2Wuji pill was better than NO.1.ConclusionThe two compatibilities of Wuji pill had definite effects on colonic motility of post inflammation irritable bowel syndrome and its mechanism is probably via bi-directional regulation of brain-gut axis, then affecting the level of5-HT, the expression of5-HT receptor3&4, the content of cAMP, PKA, Ca2+in colon and brain as well as the phosphorylational level of CREB, the quantity and condition of MC and EC in colon. |
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